Human papillomaviruses (HPV) replicate in stratified epithelia of the skin and mucosa and require the terminal differentiation program to complete their lifecycle. HPV access the basal cells of these epithelia through lesions and establish infection after genome delivery to the nucleus and initial genome amplification. In the basal cell compartment of productive infections, genome levels are maintained and early viral transcript levels are low. Early, intermediate and late transcripts as well as genome levels increase when infected cells enter the terminal differentiation program. The E6 and E7 oncoproteins prevent differentiated cells from exiting the cell cycle, resulting in the appearance of the benign lesions typically associated with HPV infection (warts, papillomas) and allowing genome amplification, late gene expression and virus production. However, some HPV types including HPV16 are associated with malignancies such as cervical carcinoma. HPV-induced transformation is initiated with the deregulation of oncogene expression in the long-lived, replication-competent cells of the basal layer. While we do have a very good understanding of E6 and E7 function during transformation, the lack of appropriate cell culture models have prevented us from studying immediate early events following infection of basal cells. Most studies of the HPV life cycle depend on keratinocytes-derived cell lines established from low-grade lesions or after transfection of viral genome into primary keratinocytes and outgrowth of HPV DNA-containing cell clones. In contrast to basal cells in productive lesions, HPV- immortalized cell lines express high levels of the E6 and E7 oncoproteins, which is a requirement for immortalization. The study of such cell lines allowed an understanding of differentiation-induced changes to viral gene expression, genome amplification and the role early viral proteins play in this process. However, many questions regarding early events during the establishment of HPV infection as well as early events of viral transformation remain unanswered. Based on our intimate studies of attachment, binding and internalization of HPV16 virions by keratinocytes and our capability to generate virions using heterologous expression systems, we have now established an infection model that allows efficient infection of primary human foreskin keratinocytes (HFK) with HPV16 quasivirions. The model mimics natural infection in that (i) it utilizes prebinding of virions to extracellular matrix, the basement membrane-equivalent; (ii) allows efficient delivery of viral genome to PML nuclear bodies, (iii) only the early but not the late promoter is active in undifferentiated HFK; (iv) early and late promoter are responsive to differentiation triggered by growth in methylcellulose or organotypic raft cultures resulting in high levels of late transcripts; (v) viral genome remains episomal and is amplified upon differentiation; (vi) and capsid proteins are expressed in the upper layers of organotypic raft cultures derived from HPV16-infected HFK. Thus, the infection model is the first cell culture model to recapitulate the complete viral lifecycle. Since the early promoter is upregulated upon differentiation, we assume that the infection model also mimics early promoter repression observed in naturally infected lesions. We have gathered preliminary evidence using next generation RNA sequencing that only a very limited subset of host cell genes is differentially expressed in HPV16-infected HFK as opposed to thousands of genes in HPV16-immortalized HFK. Because the infection model uses quasivirions generated in the 293TT production cell line, which does not depend on any non- structural HPV factor for virus production, it is amenable to extensive genetic manipulations. In turn, this allows the characterization of viral factors involved in the immediate early events of HPV16 infection. We have prove- of-principal that mutant viruses can be generated and found evidence that E7 knockout affects early and late viral promoter activity in monolayer and differentiated HFK cells, respectively. We propose to utilize the infection model to determine the role of viral and host cell factors in genome amplification and regulation of early promoter activity (Aim1); delineate the contributions of E6 and E7 to the HPV16 lifecycle (Aim 2); and compare the contributions of E6 and E7 to the lifecycles of low- and high-risk HPV types (Aim 3). The proposed studies will help fill huge gaps in the understanding of immediate early events of the HPV lifecycle. They will also help to gain a better understanding of the often hypothesized but never experimentally tested repression of oncogene expression in the basal cell layer during natural infection and the role viral proteins play during this process. In future, this model will allow investigating the deregulation of viral oncogene expression during initial events of transformation. The infection model has the potential to be as important for the study of immediate early events of the HPV16 lifecycle and transformation as the establishment of HPV- harboring keratinocytes was for understanding the late differentiation-induced stages.